CN112646908A - Vibrio vulnificus isothermal amplification primer, probe, kit and detection method - Google Patents
Vibrio vulnificus isothermal amplification primer, probe, kit and detection method Download PDFInfo
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Abstract
The invention discloses a vibrio vulnificus isothermal amplification primer, which comprises an upstream primer and a downstream primer; the sequence of the upstream primer is SEQ ID NO: 1; the sequence of the downstream primer is SEQ ID NO: 2. the primer and the probe provided by the invention have good specificity, and the provided kit can improve the specificity by specifically enriching a target sequence through a target capture probe; meanwhile, the capture probe contains a T7 promoter sequence, and a sequence to be detected is amplified through transcription and reverse transcription reaction, so that the detection sensitivity can be greatly improved, the operation is simple, convenient and quick, the problem of inhibiting the isothermal amplification of the recombinase by a complex sample background can be solved, the practical application problem of the isothermal amplification of the recombinase is solved, and the method has a wide application scene.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to vibrio vulnificus isothermal amplification primers, probes, a kit and a detection method.
Background
Vibrio vulnificus (Vibrio vulgaris) is a bacterium which generally lives in the sea, and once infected, the disease is acute, the disease condition is rapidly developed, and the Vibrio vulnificus, Vibrio cholerae and Vibrio enteritis are listed as three kinds of Vibrio which cause human infectious diseases. Sea products or seawater polluted by vibrio vulnificus or sea fish and shellfish which are stabbed by the vibrio vulnificus can cause the vibrio vulnificus to enter a human body and cause infection. Symptoms after vibrio vulnificus infection comprise vomit, fever, diarrhea, hypotension, swelling, pain and the like, clinically serious septicemia and limb necrosis are easily caused, and the treatment with antibiotics is needed as soon as possible.
Isothermal amplification techniques, also known as isothermal amplification techniques, can amplify specific DNA or RNA fragments at a constant temperature. Compared with the conventional PCR amplification technology, the isothermal amplification technology has low requirements on instruments and short reaction time, and can better meet the requirements of quick real-time detection of POCT. The existing isothermal amplification technology mainly comprises loop-mediated isothermal amplification (LAMP), nucleic acid sequence dependent amplification (NASBA), Rolling Circle Amplification (RCA), helicase isothermal amplification (HAD) and the like, and is widely applied to the fields of food safety, public health and epidemic prevention and the like. Although isothermal amplification techniques have many advantages, due to the complexity of the sample background, the techniques such as isothermal amplification of recombinant enzymes are easily inhibited, and thus the practical application of the techniques such as isothermal amplification of recombinant enzymes is limited.
The harm of infected vibrio vulnificus is large, and the prior art cannot detect the vibrio vulnificus quickly and efficiently, so that the provision of the vibrio vulnificus isothermal amplification primer, the probe, the kit and the detection method have important significance.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a vibrio vulnificus isothermal amplification primer, a probe, a kit and a detection method, the provided primer and probe have good specificity, and the rapid and efficient detection of vibrio vulnificus is realized by combining a recombinase isothermal amplification technology. In addition, the kit provided by the invention specifically enriches a target sequence through a target capture probe, so that the specificity can be improved; meanwhile, the capture probe contains a T7 promoter sequence, and a sequence to be detected is amplified through transcription and reverse transcription reaction, so that the detection sensitivity can be greatly improved, the operation is simple, convenient and quick, the problem of inhibiting the isothermal amplification of the recombinase by a complex sample background can be solved, the practical application problem of the isothermal amplification of the recombinase is solved, and the method has a wide application scene.
The objects of the invention will be further illustrated by the following detailed description.
The invention provides vibrio vulnificus isothermal amplification primers, which comprise an upstream primer and a downstream primer; the sequence of the upstream primer (primer F) is CACCGCCGCTCACTGGGGCAGTGGCTGGGTAT (SEQ ID NO: 1); the sequence of the downstream primer (primer R) is CGATAGTTGAGTTTCACGCCCATCTCGAAATCCG (SEQ ID NO: 2).
The invention also provides a vibrio vulnificus isothermal amplification probe, which is used for detecting the amplification product of the vibrio vulnificus isothermal amplification primer in claim 1, the sequence of the probe is modified by TCTTATTCCAACTTCAAACCGAACTATGACGTTGACGAAGCGCCTGTG (SEQ ID NO: 3), and the modified probe has the sequence: TCTTATTCCAACTTCAAACCGAACTATGACGT/i6FAMdT/T/idSp/G/iBHQ1dT/ACGAAGCGCCTGTG C3 Spacer.
In addition, the vibrio vulnificus isothermal amplification kit is characterized in that: comprises the vibrio vulnificus isothermal amplification primer and the vibrio vulnificus isothermal amplification probe.
Preferably, the concentration of the vibrio vulnificus isothermal amplification primer is 5-20 mu mol/L, and the concentration of the vibrio vulnificus isothermal amplification probe is 5-20 mu mol/L.
Preferably, the vibrio vulnificus isothermal amplification kit further comprises capture magnetic beads, an amplification reaction solution and a recombinase.
The kit provided by the invention adopts the combination of the magnetic bead targeted capture probe and recombinase isothermal amplification, and can reduce the influence of background genes on amplificationThe amplification accuracy is ensured, the specificity is improved, and the operation is simple and convenient; the capture probe carries a promoter, and can obtain a large number of target sequences through RNA polymerase and reverse transcriptase after being combined with the target sequences, so that the detection sensitivity is improved. The capture beads carry a capture probe (underlined T7 promoter sequence) NH2C6-TAATACGACTCA CTATAGGGCCAGTCGATGCGAATACGTTGTTTCACGGT。
More preferably, the vibrio vulnificus isothermal amplification kit further comprises a sample lysate and a positive control.
Correspondingly, the invention also provides a vibrio vulnificus isothermal amplification detection method, which comprises the following steps:
s1, preprocessing a sample, extracting DNA of the sample, and performing targeted enrichment on the sample by using the capture magnetic beads to obtain the capture magnetic beads after targeted enrichment;
s2, establishing an amplification system comprising the vibrio vulnificus isothermal amplification primer, the vibrio vulnificus isothermal amplification probe and a recombinase, adding the targeted enriched capture magnetic beads, and reacting the steps and conditions comprising: 1) preheating: cycling at 40 ℃ for 1s and 1 cycle; 2) extension and fluorescence collection: and (3) carrying out 30s and 30 cycles at 40 ℃, carrying out constant-temperature amplification to detect fluorescence, collecting a fluorescence signal, and judging a sample detection result.
By specifically enriching the target sequence, the influence of a complex sample background on the isothermal amplification inhibition of the recombinant enzyme can be avoided, and the specificity and the accuracy can be improved.
Preferably, the targeted enrichment of the sample comprises the steps of: adding the capture magnetic beads, uniformly mixing by vortex, and incubating for 5-15 min at 90-95 ℃. Centrifuging, adsorbing by a magnetic frame, and discarding the waste liquid. Washing with nuclease-free deionized water, and adding nuclease-free deionized water as a nucleic acid preservation solution to obtain the target-enriched capture magnetic beads.
Compared with the prior art, the invention has the beneficial effects that:
(1) the invention downloads Vibrio vulnificus specific conserved gene vvhA gene sequence (MH357338.1) through the Genebank of NCBI, designs primers and probes after comparison, and performs screening and optimization, thereby having good specificity.
(2) The invention provides a vibrio vulnificus isothermal amplification kit and a detection method, wherein optimized primers and probes are used, a magnetic bead targeted capture probe is combined with recombinase isothermal amplification, rapid amplification is carried out under a constant temperature condition, an amplification experiment is completed within 10min, the influence of background genes on amplification can be reduced, the amplification accuracy is ensured, the specificity is improved, the operation is simple and convenient, the detection sensitivity is high, the application range is wide, and the kit and the detection method have important significance for realizing the rapid detection application of an isothermal amplification technology in basic medical health institutions, improving the sanitation and epidemic prevention level and the like.
Drawings
FIG. 1 is a schematic diagram of the detection method of the present invention.
FIG. 2 is a diagram showing the results of the combination of primers and probe screening for Vibrio vulnificus.
FIG. 3 is a graph showing the results of the sensitivity detection of the present invention.
FIG. 4 is a schematic diagram showing the results of the specific detection according to the present invention.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples.
In the present invention, the reagents and materials are all conventional commercially available products or can be obtained by means of conventional techniques in the art. The carboxyl magnetic beads are purchased from Suzhou beaver biomedical engineering Co., Ltd, and the vibrio vulnificus is from the Guangdong province microorganism strain preservation center. The% concentration in the present invention, if not explicitly indicated, refers to mass concentration, or as commonly understood in the industry.
EXAMPLE one preparation of Capture magnetic beads
The preparation system of the capture magnetic beads is as follows:
the preparation method of the capture magnetic beads comprises the following steps:
(1) after the preparation system is prepared, uniformly mixing by vortex, incubating for 4h at 37 ℃, and uniformly mixing by vortex for 1 time every 30 min;
(2) after the reaction is finished, centrifugally collecting magnetic beads on the tube cover and the wall, placing the tube cover and the wall on a magnetic frame, and discarding the supernatant;
(3) 500 μ L of 1mol/L NaHCO was added3Washing for 2 times at room temperature;
(4) 500 μ L of 1mol/L NaHCO was added3Incubating for 30 min;
(5) adding 500 mu L of nuclease-free water, washing for 1 time at room temperature, and discarding the waste liquid;
(6) add 500. mu.L of 0.01% Proclin resuspended beads to obtain captured beads, and store at 4 ℃.
EXAMPLE two sample Targeted enrichment
Performing sample pretreatment, extracting sample DNA, adding the capture magnetic beads prepared in the first embodiment, performing vortex mixing, incubating at 92 ℃ for 10min, centrifuging, adsorbing for 3min by a magnetic frame, discarding waste liquid, washing by using nuclease-free deionized water, and adding nuclease-free deionized water as a nucleic acid preservation solution to obtain the target-enriched capture magnetic beads, wherein the mass concentration of the target-enriched capture magnetic beads is 1%.
Example Triplex enzyme isothermal amplification assay
An amplification system: 60mmol/L Tris (pH 7.2), 6mmol/L dithiothreitol, 5% polyethylene glycol, 5mmol/L ATP, 1.5mmol/L dNTPs, 100. mu. mol/L phosphoinositide, 50 ng/. mu.L single-stranded binding protein SSB, 10 ng/. mu.L RecQ helicase, 50 ng/. mu.L UvsX recombinase, 50 ng/. mu.L UvsY recombinase, 70 ng/. mu.L BSU DNA polymerase, 10U RNase H, 1U exonuclease III, 1. mu. mol/L primer F, 1. mu. mol/L primer R, 0.8. mu. mol/L modified probe, 20mmol/L magnesium acetate. After the preparation, 35. mu.L of the amplification system was taken and 15. mu.L of the targeted and enriched capture magnetic beads was added.
And (3) detection procedures:
| step (ii) of | Procedure | Temperature of | Time | Number of |
|
| 1 | |
40 | 1s | 1 | |
| 2 | Extension and |
40 | 30s | 40 |
And automatically storing the result after the reaction is finished. Results were analyzed and determined as in table 1 using the raw curve and endpoint fluorescence values, without setting baseline and threshold values. The schematic diagram of the detection method provided by the invention is shown in FIG. 1.
TABLE 1 analysis and determination of results
Example four Vibrio vulnificus isothermal amplification primers and Probe design and optimization
Synthesizing a VVHA gene sequence (TTTGGTGAGTGTGATGAACTGCGCCGCCAAGAGCTTGGATGCTATTTCACCGCCGCTCACTGGGGCAGTGGCTGGGTATTTGATAAGACGAAGTTCAACCCAATCTCTTATTCCAACTTCAAACCGAACTATGACGTTTTGTACGAAGCGCCTGTGTCTGAAACTGGCGTAACGGATTTCGAGATGGGCGTGAAACTCAACTATCGTGCACGCTTTGGTACGGTAATCCCTTCAGCACTCTTCTCAGTTTATGGTTCTGCGGGCTCGTCAACCAACAGCAGTACCGTGAAACAACGTATTCGCATCGACTGGAATCATCCACT) of the vibrio vulnificus specific conserved gene, connecting the VVHA gene sequence into a pUC57 plasmid, and detecting by using plasmid DNA by using designed primers, probes and isothermal amplification reaction liquid of the vibrio vulnificus. The primers synthesized for the total of 9 combinations were combined as shown in Table 2, and one combination with the best amplification curve was selected. As can be seen from fig. 2: the primer probe combinations vvF2, vvR1 and vvP1 gave the best amplification results, with the smoothest curve morphology.
TABLE 2 primer and probe screening combinations for Vibrio vulnificus
vvF1:CAGTGGCTGGGTATTTGATAAGACGAAGTTCAAC；
vvF2:CACCGCCGCTCACTGGGGCAGTGGCTGGGTAT；
vvF3:CAAGAGCTTGGATGCTATTTCACCGCCGCTCAC；
vvR1:CGATAGTTGAGTTTCACGCCCATCTCGAAATCCG；
vvR2:CGTACCAAAGCGTGCACGATAGTTGAGTTTCAC；
vvR3:GCTGAAGGGATTACCGTACCAAAGCGTGCACGATA；
Fluorescent probe vvP 1:
TCTTATTCCAACTTCAAACCGAACTATGACGT/i6FAMdT/T/idSp/G/iBHQ1dT/ACGAAGCGCCTGTG3'Spacer C3。
EXAMPLE four sensitivity and specificity of detection
Sending Vibrio vulnificus to Shenzhen Longhua disease prevention and control center, entrusting the Vibrio vulnificus to carry out strain culture, colony counting and nucleic acid extraction of enrichment fluid with different concentrations, and carrying out DNA extraction of the enrichment fluid with different concentrations by adopting a water boiling method to extract 9 concentrations in total. Wherein the concentration of the bacteria diluent is 10-4And 10-5The colony plate coating count was performed, 100ul of the corresponding bacteria solution was applied to each plate, 3 plates were coated at each concentration, and the colony count results are shown in table 3.
TABLE 3 Vibrio vulnificus colony counts
According to the colony count of Shenzhen Longhua disease prevention and control center, 10 should be selected according to the requirements of 'Chinese animal pharmacopoeia' 2015 edition on colony count-4As an ideal counting result. Calculating the concentration of enrichment liquid with different concentrations as follows:
initial concentration: 1.84X 107cfu/ml
1：1.84×105CFU/ml
2：1.84×104CFU/ml
3：1.84×103CFU/ml
4：1.84×102CFU/ml
5：1.84×101CFU/ml
6：1.84×100CFU/ml
The sensitivity detection result of the invention is shown in figure 3, and the lowest detection limit of the kit can reach 1.84 multiplied by 102CFU/ml, the reaction time can be completed within 10 min.
Performing specificity detection with Staphylococcus aureus, Pseudomonas aeruginosa, Clostridium perfringens, Bacillus cereus, Streptococcus agalactiae, enterococcus casseliflavus, Streptococcus pneumoniae, Escherichia coli, and Klebsiella pneumoniae at a concentration of 106CFU/ml, the results are shown in FIG. 4, which shows that the detection specificity of the invention is good, and other bacteria such as staphylococcus aureus can not cause interference.
The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.
Sequence listing
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Claims (9)
1. The vibrio vulnificus isothermal amplification primer is characterized by comprising the following components in parts by weight: comprises an upstream primer and a downstream primer; the sequence of the upstream primer is SEQ ID NO: 1; the sequence of the downstream primer is SEQ ID NO: 2.
2. the vibrio vulnificus isothermal amplification probe is characterized in that: the probe is used for detecting the amplification product of the vibrio vulnificus isothermal amplification primer of claim 1, and the sequence of the probe is represented by SEQ ID NO: 3, the modified probe sequence is as follows: TCTTATTCCAACTTCAAACCGAACTATGACGT/i6FAMdT/T/idSp/G/iBHQ1dT/ACGAAGCGCCTGTG3' Spacer C3.
3. The vibrio vulnificus isothermal amplification kit is characterized in that: comprises the Vibrio vulnificus isothermal amplification primer of claim 1 and the Vibrio vulnificus isothermal amplification probe of claim 2.
4. The Vibrio vulnificus isothermal amplification kit according to claim 3, wherein: the concentration of the vibrio vulnificus isothermal amplification primer is 5-20 mu mol/L, and the concentration of the vibrio vulnificus isothermal amplification probe is 5-20 mu mol/L.
5. The isothermal amplification kit of Vibrio vulnificus according to claim 3 or 4, wherein: the kit also comprises capture magnetic beads, an amplification reaction solution and a recombinase.
6. The Vibrio vulnificus isothermal amplification kit according to claim 5, wherein: the capture magnetic bead is provided with a capture probe NH2C6-TAATACGACTCACTATAGGGCCAGTCGATGCGAATACGTTGTTTCACGGT。
7. The isothermal amplification kit for Vibrio vulnificus according to claim 5 or 6, wherein: also included are sample lysates and positive controls.
8. The vibrio vulnificus isothermal amplification detection method is characterized by comprising the following steps of: the method comprises the following steps:
s1, preprocessing a sample, extracting DNA of the sample, and performing targeted enrichment on the sample by using the capture magnetic beads to obtain the capture magnetic beads after targeted enrichment;
s2, establishing an amplification system comprising the vibrio vulnificus isothermal amplification primer of claim 1, the vibrio vulnificus isothermal amplification probe of claim 2 and a recombinase, adding the target-enriched capture magnetic beads, and reacting steps and conditions comprising: 1) preheating: cycling at 40 ℃ for 1s and 1 cycle; 2) extension and fluorescence collection: and (3) carrying out 30s and 30 cycles at 40 ℃, carrying out constant-temperature amplification to detect fluorescence, collecting a fluorescence signal, and judging a sample detection result.
9. The method for isothermal amplification detection of Vibrio vulnificus according to claim 8, wherein: the targeted enrichment of the sample comprises the following steps: adding capture magnetic beads, performing vortex mixing, incubating for 5-15 min at 90-95 ℃, centrifuging, adsorbing by a magnetic frame, removing waste liquid, collecting the capture magnetic beads, adding nuclease-free deionized water for washing, and adding nuclease-free deionized water as a nucleic acid preservation solution to obtain the target-enriched capture magnetic beads.
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